【作者】 雷钧；

【导师】 陈知水；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的：研究Th17细胞及其相关细胞因子IL-17在小鼠心脏移植急性排斥反应中的表达及意义。方法：建立小鼠颈部异位心脏移植模型,C57小鼠作为受体,BALB/c小鼠作为供体或受体,实验动物随机分为同系移植组和同种异体移植组,观察2组移植心脏的存活时间。应用realtime-PCR检测各组移植心脏中IL-17、RORγt、IFN-γmRNA在移植术后第1天至第7天的动态表达水平；Western blot检测各组移植心脏中IL-17、RORγt、IFN-γ在移植术后第1、3、5、7天的表达水平；HE染色检测移植术后第7天各组移植物中淋巴细胞浸润情况；免疫荧光法观察同种异体移植组术后第7天移植心脏中CD4+、CD8+T细胞的浸润情况,并检测CD4+、CD8+T细胞中细胞因子IL-17的表达。结果：同种异体移植组中,IL-17和RORγtmRNA的表达均早于IFN-γ,在移植术后第1天即可于移植心脏中检测到两者的表达,而IFN-γmRNA在移植术后第2天才可检测到表达,三者mRNA的转录水平伴随移植排斥反应的进程均逐渐升高。Western blot检测各组移植物中IL-17、RORγt、IFN-γ蛋白表达亦得出相似的结果。免疫荧光结果显示,移植术后第7天同种异体移植组移植心脏中有大量CD4+、CD8+T细胞浸润,而细胞因子IL-17主要由CD4+T细胞即Th17细胞分泌。结论：Th17细胞参与了小鼠同种异体心脏移植急性排斥反应的发生和发展,对移植脏器中细胞因子IL-17的检测可作为急性排斥反应早期诊断的预见性和特异性指标。目的：观察应用anti-IL-6 mAb对同种异体小鼠心脏移植急性排斥反应的抑制作用,并探讨其可能的作用机制。方法：以Balb/c和C57小鼠分别作为供受体,建立小鼠心脏移植急性排斥反应模型。实验动物随机分为anti-IL-6 mAb组、anti-IL-17 mAb组及rat-IgG对照组,术后每日通过触诊受鼠颈部移植心脏观察各组小鼠移植心脏存活时间并结合组织学检测。应用realtime-PCR检测各组移植心脏中细胞因子IL-17、IFN-γ及Th17细胞特异性转录因子RORγtmRNA表达水平,流式细胞术分别检测各组受鼠脾脏CD4+、CD8+T细胞中IL-17、IFN-γ的表达。结果：Anti-IL-6 mAb、anti-IL-17 mAb输注均可显著抑制小鼠急性排斥反应的发生,移植心脏存活时间分别延长至(22.0±2.3天)、(13±1.8天),anti-IL-6 mAb组抑制排斥反应发生的效果更加显著。组织病理学结果显示anti-IL-6 mAb输注可减轻移植心肌结构的破坏,移植心脏中浸润的淋巴细胞明显减少。Realtime-PCR结果显示,anti-IL-6 mAb组移植心脏中IL-17、IFN-γ和RORγtmRNA的转录水平较rat-IgG对照组及未治疗组显著下降,anti-IL-17 mAb输注可部分下调IFN-γmRNA的转录水平。流式细胞仪检测结果显示anti-IL-6 mAb组受鼠脾脏CD4+及CD8+T细胞中IFN-γ的比例均有不同程度的下降,且CD4+IFN-γ+T细胞较CD8+IFN-γ+T细胞下降更为明显,同时CD4+IL-17+T(Th17)细胞的比例在anti-IL-6 mAb组中亦明显下降。结论：anti-IL-6 mAb可有效抑制急性排斥反应小鼠体内Th17和Th1细胞的活化,并延长同种异体心脏移植物的存活时间。目的：探讨CD4+CD25+Foxp3+调节性T细胞在anti-IL-6 mAb抑制同种异体心脏移植急性排斥反应中的作用及机制。方法：以Balb/c和C57BL/6小鼠分别作为供受体,建立小鼠心脏移植急性排斥反应模型。实验动物随机分为anti-IL-6 mAb组、anti-IL-17mAb组及rat-IgG对照组,流式细胞仪检测各组受鼠移植术后第7天脾脏及移植心脏中CD4+CD25+Foxp3+调节性T细胞的比例。分别于anti-IL-6 mAb组术后第1、3、5天给予anti-CD25 mAb输注,于术后第7天取受鼠脾脏及移植心脏检测CD4+CD25+Foxp3+调节性T细胞的比例,观察移植心脏存活时间并结合组织学检查；realtime-PCR检测anti-CD25 mAb输注后第3、7、10天移植心脏中IFN-γmRNA的表达情况。使用去增殖的BALB/c或C3H小鼠脾脏淋巴细胞作为刺激细胞,各实验组受鼠移植物来源的淋巴细胞作为反应细胞,行混合淋巴细胞培养检测细胞增殖情况。结果：Anti-IL-6 mAb组移植术后第7天CD4+CD25+T细胞的比例明显升高,CD4+CD25+Foxp3+调节性T细胞占CD3+T细胞的比例表现出同样的趋势,而anti-IL-17mAb组与未治疗组比较无明显差异。Anti-CD25 mAb输注anti-IL-6 mAb组明显缩短了小鼠移植心脏的存活时间(10.6±1.2天),伴随着CD4+CD25+Foxp3+调节性T细胞占CD3+T细胞的比例明显下降,同时移植物中IFN-γmRNA的转录水平升高。混合淋巴细胞培养结果显示anti-IL-6 mAb组受鼠淋巴细胞经刺激后其细胞增殖程度较rat-IgG对照组、anti-IL-6 mAb+anti-CD25 mAb组及未治疗组明显下降,而针对来自第三方C3H小鼠的脾脏淋巴细胞刺激则无此抑制效应。结论：Anti-IL-6 mAb延长小鼠移植心脏存活可能是通过促进移植小鼠脾脏及移植心脏中CD4+CD25+Foxp3+调节性T细胞的产生而实现的。使用anti-CD25 mAb降低CD4+CD25+T细胞水平抑制了anti-IL-6 mAb的抗排斥效应,并且伴随着移植物中IFN-γmRNA转录水平的升高。更多还原

【Abstract】 Objective:To investigate the role of Th17 cell and its cytokines on cardiac acute allograft rejection in mice.Methods:The heterotopic cardiac transplantation models was established, C57BL/6 mice were used as recipients, whereas BALB/c mice were used as donors, recipients mice were randomly divided into two groups:isograft group and untreated allogeneic group. Mean survival time (MST) was measured. The grafts of recipient in each group were harvested on postoperative day (POD) 1 to 7. The expression of IL-17、RORγt and IFN-γwere detected by realtime-PCR and Western blot. The expression of IL-17 in CD4+or CD8+T cells were observed on POD 7 by immunofluorescence techniques.Results:The examination of allograft cytokines mRNA showed that IL-17 and RORyt mRNA expressed earlier on the first postoperative day. However we could not find the same early appearance on IFN-γ. Allograft expression for IL-17, RORyt and IFN-γincreased significantly in untreated allogeneic group throughout the experimental time. Immunofluorescence staining of the allografts indicated that both CD4+and CD8+T cells were present on POD 7, but the majority of the graft-infiltrating lymphocytes in untreated allogeneic group were IL-17-producing CD4+T cells.Conclusion:Th17 cells may play an important role in the development of cardiac transplant rejection. IL-17 could serve as a predictive parameter for allograft rejection in the future.Objective:To investigate the effects of anti-IL-6 mAb on acute allograft rejection and the potential mechanisms in a mouse heart transplantation model.Methods:The heterotopic cardiac transplantation models was performed from Balb/c to C57 mice. The anti-IL-6 mAb was administered to recipient mice after cardiac grafting. Results were compared with administration of anti-IL-17 mAb or rat-IgG The strength and quality of cardiac impulses were assessed by palpation on daily basis in combination of histological evaluation of graft. Quantitative polymerase chain reaction assay and flow cytometric analysis were employed to determine the mRNA expression of pro-inflammatory cytokines in graft-infiltrating lymphocytes and splenocytes of recipients, respectively.Results:The results showed that the cardiac allograft survival in anti-IL-6 mAb treated mice prolonged significantly compared to that of the untreated or anti-IL-17 mAb treated mice (22.0±2.3 d, P<0.01). Histological analysis of cardiac allografts harvested on POD 7 in anti-IL-6 mAb treated mice revealed relatively intact myocardium and remarkable reduced infiltration of inflammatory cells. The mRNA expression of IL-17、IFN-γand RORγt in heart treated with anti-IL-6 mAb was markedly reduced. Whereas administration of anti-IL-17 mAb only modestly attenuated the mRNA expression of IFN-γ. Anti-IL-6 mAb treatment suppressed the production of IFN-γin both CD4+and CD8+T cells. The proportion of CD4+IFN-γ+cells in the recipients treated with anti-IL-6 mAb decreased significantly than those mice treated with anti-IL-17 mAb or rat-IgG or untreated. The population of T cells expressing IFN-γreduced more markly for CD4+T cells than for CD8+T cells. Moreover, compared with untreated mice, the proportion of CD4+IL-17+ cells (Th17) in anti-IL-6 mAb treated was dramatically decreased, whereas slightly reduced IL-17-producing CD8+T cells in the spleen.Conclusion:Administration with anti-IL-6 mAb may be protective against acute rejection after cardiac transplantation through suppressing the activation of effector Th17 and Thl cells.Objective:To assess the proportion and function of CD4+CD25+Foxp3+regulatory T cells in anti-IL-6 mAb treated mice.Methods:The heterotopic cardiac transplantation models was performed from Balb/c to C57 mice. The anti-IL-6 mAb or anti-IL-17 mAb or rat-IgG was administered to recipient mice after cardiac grafting. The proportion of CD4+CD25+Foxp3+Treg cells were determined in the allograft and spleen in POD 7 by flow cytometry. Anti-CD25 mAb was administered on POD 1,3, and 5 to delete CD4+CD25+Foxp3+Treg cells in anti-IL-6 mAb treated mice and graft survivals were compared. Allograft of recipients were collected on POD 3,7 and 10, then subjected to expression analysis of IFN-γby real-time PCR. Mitomycin-treated BALB/c or C3H splenocytes were used as stimulator cells, T cells of GIL isolated from each treatment recipients on POD 5 were used as responder cells, and lymphocytes proliferations were observed.Results:The proportion of CD4+CD25+T cells increased in both spleen and allografts pre-treated with anti-IL-6 mAb, moreover, the average percentage of CD4+CD25+Foxp3+ Cells in the CD3+T-cell pool showed significant increase on POD 7 after anti-IL-6 mAb administration in the spleen and allografts compared to those recipients pre-treated with rat-IgG or untreated. Whereas the anti-IL-17 mAb treated mice have no evident increased proportion of Tregs. Administration of anti-CD25 mAb to anti-IL-6 mAb treated mice decreased the absolute number of CD4+CD25+Foxp3+Tregs. The MST of grafts in anti-IL-6 mAb+anti-CD25 mAb treated mice was 10.6±1.2 d, which was slightly longer than untreated mice (P>0.05). Moreover, anti-IL-6 mAb treated mice receiving anti-CD25 mAb displayed restored the expression of IFN-y when compared with mice treated with anti-IL-6 mAb alone as determined by real-time PCR. In comparison with GIL from untreated mice, GIL from allograft of anti-IL-6 mAb treated mice showed a profound inhibition of proliferation in response to donor cells, but not to third-party cells.Conclusion:Increased CD4+CD25+Foxp3+Treg cells play an important role in anti-IL-6 mAb treated mice, at least partly via decreasing the expression of IFN-γ.更多还原